The importance of FOXP3 in the development and function of nTregs is quite clear. However, the underlying molecular mechanism by which FOXP3 functions remains to be elucidated. FOXP3 has 3 discernible functional domains, a carboxyl-termination forkhead domain (FKH; a.a. 338–421), a single C2H2 zinc finger (a.a. 200–223) and a leucine zipper-like motif (a.a. 240–261). There is also a domain at the amino terminal region that is somewhat Proline-rich (a.a. 1–193). Some clues as to the function of these domains have come from an analysis of FOXP3 variants containing mutations found in IPEX patients. These patients, as described above, develop a variety of symptoms consistent with a Treg deficit10
. Mutations have been found throughout FOXP3, including in each of the domains described above except for the zinc-finger, demonstrating that these regions of the protein as important for proper function10;11
. IPEX mutations in the FKH domain have been shown to affect DNA binding, while 2 separate mutations in the leucine zipper have been found to affect FOXP3 homo- and hetero-dimerization12–14
. Further evidence for the importance of the Leucine zipper domain comes from studies of the related FoxP1 and P2, where deletion of this domain abrogated their ability to act as transcriptional repressors14;15
. Finally, 3 missense mutations have been found in the amino-terminal domain; the affect of these mutations on FOXP3 function is currently underway.
Site directed mutagenesis has also been used to further define FOXP3 function. Using a GAL4-FOXP3 fusion protein and a reporter construct consisting of ARRE2- and GAL4-binding sites, we have begun to define the regions of FOXP3 that are required for transcriptional regulation13
. This study showed that the amino terminal 198 amino acids of FOXP3 contains all the sequences required to inhibit transcriptional activation by NFAT; however, it should be noted that the DNA-binding domain for GAL4 contains both nuclear localization and dimerization motifs. Consistent with these studies, Bettelli et al. showed that the amino terminal region of FOXP3 was important for inhibiting transcription mediated by NFAT (nuclear factor of activated T cells)16
. Recently, Wu et al. extended these studies by showing that FOXP3 and NFAT can bind cooperatively to the ARRE2 site on the IL-2 promoter17
. Mutation of the residues in the FKH domain predicted to interact with NFAT abolished its ability to inhibit transcription. Taken as a whole, these data suggest that at least one mechanism of FOXP3-mediated transcriptional repression involves direct contact with NFAT, and subsequent inhibition.
Recent work has begun to define the nature of the protein complexes that associate with FOXP3. Initial studies focused on other transcription factors whose activity was inhibited by FOXP3, including NFAT and NFκB. Both NFAT and NFκB were found to be capable of co-immunoprecipitating with FOXP318
, and subsequent studies showed that FOXP3 and NFAT bound cooperatively to the IL-2 promoter17
. In the latter study, based on structural analyses, several residues in the forkhead domain of FOXP3 were predicted to interact directly with NFAT, and subsequent mutagenesis of these residues showed a decrease in the ability of FOXP3 to inhibit IL-2 production 17
. More recently, Lee et al. have shown that FOXP3 can interact with phosphorylated c-Jun, and thereby alter its subnuclear localization and inhibit AP-1 DNA binding19
. Finally, FOXP3 has also been shown capable of interacting with Runx1/AML120
. Runx family members (runx1, 2, and 3) are critical for hematopoietic development, and perform a variety of functions in CD4 T cells21;22
. Ono et al. found that FOXP3 and Runx1 interacted on the IL-2 promoter in a manner different from FOXP3-NFAT in two ways20
. First, the interacting sites were no in either DNA binding domain. Second, the binding sites for each transcription factor on the promoter were physically distant. This suggests the possibility of a tri-partite complex involving FOXP3, NFAT, and Runx1 on the IL-2 promoter.
Using a yeast two-hybrid screening approach, Du et al used the amino terminal region of FOXP3 to screen a library generated from human TR
. Among the FOXP3-interacting proteins found in the screen was the retinoic acid receptor-like orphan receptor (ROR)-α. Further characterization showed that the interaction was both physical and functional in that FOXP3 was capable of inhibiting RORα-mediated transcriptional activation. Interestingly, the ΔE2 isoform of FOXP3 did not interact with RORα, the first demonstration of a function distinction between the two proteins. Also, the forkhead domain was not required for FOXP3 to inhibit RORα-mediated transcriptional activation, suggesting that FOXP3 is acting as a transcriptional co-repressor in this setting23
FOXP3 was found to bind to the AF2 domain of RORα (also known as helix 12). Within the steroid hormone nuclear receptor family, this domain is functionally important in that it binds transcriptional co-repressors in the absence of ligand, and following a conformational change, binds co-activators after ligand binding24;25
. Members of the ROR family have a constitutive ‘active’ confirmation, suggesting that they bind to an endogenous ligand. Consistent with this model, RORα was co-crystallized with a molecule of cholesterol in its ligand binding pocket26
. The binding motif present on the co-activators (members of the Steroid Co-Activator, or SRC, family) required for binding to the AF2 domain is LxxLL, where x is any amino acid27;28
. There is a single such motif in FOXP3, in exon 2, providing a mechanistic explanation for the failure of the ΔE2 isoform to bind to RORα.
The relative importance of each of these interactions in the overall function of FOXP3 remains to be determined. RORα has been shown to regulate inflammatory responses, so its interaction with FOXP3 may be involved in regulating responses29;30
. As described below, the interaction with ROR family members has potential consequences for peripheral CD4 T cell effector differentiation.